The mechanisms whereby cells develop resistance to the cancer chemotherapeutic agent, vincristine (VCR), are not entirely known. We have a system that will provide new information about this problem. The VCR-resistant cells, designated DC-3F/VCRd, available for the studies propsed are highly resistant to selective agent compared to the drug-sensitive Chinese hamster lung cells ("DC-3F") that serve as control. The DC-3F/VCRd cells are unique and interesting because 1) they are highly resistant to VCR (650-fold); 2) they contain a long, homogeneously staining region (HSR) similar to the HSRs of antifolate-resistant cells; 3) they have decreased permeability to VCR (and actinomycin D); 4) there are plasma membrane glycoprotein alterations as well as alterations of ganglioside biosynthesis; and 5) they exhibit a marked dimunition of tumor-producing capacity and striking alterations of morphology and growth behavior compared to DC-3F. We will incorporate our previous findings of resistance-related changes in DC-3F/VCRd cells in a newly-directed program of study of molecular mechanisms of VCR resistance. The selection of a VCR-resistant phenotype with an HSR compellingly leads us to study increased production of a protein associated with cytoskeletal structures, particularly with microtubules to which VCR binds specifically. Specific aims include: 1) comparison of major cytoskeletal and membrane proteins in control and VCR-resistant cells by SDS-PAGE and 2D gel electrophoresis; 2) Investigation of microtubule protein changes in DC-3F/VCRd cells and characterization of two proteins, 22,000 and 61,000 daltons respectively, revealed on gels of DC-3F/VCRd cytosols; 3) in vitro translation of polysomal RNA isolated from resistant and control cells to identify any increased transcription of specific mRNAs; 4) in situ hybridization to metaphase chromosomes of cDNA probe to total mRNA isolated from VCR-resistant cells to provide evidence for possible transcriptional activity of the HSR; 5) comparative study of influx and efflux of VCR in VCR-resistant and control cells; 6) characterization of a 150,000 dalton plasma glycoprotein selectivity increased in VCR-resistant cells compared to DC-3F cells.